Role Of Plasmodium Vivax Exported Protein PvEXP100 In Host Immune Evasion And Its Application In Cancer Therapy

Malaria is one of the most serious public health problems in the world.Plasmodium exists and proliferates even under the conditions of acquired immunity developed after malaria infection,which is called Plasmodium immune evasion.The immune protection caused by immune evasion is unstable and the specific mechanism is unknown,which hinders the basic research of malaria and the development of malaria vaccine.Therefore,it is very important to study the immune escape mechanism for the Plasmodium prevention and treatment of malaria.Studies have shown that Plasmodium export proteins play an important role in immune evasion.Exported protein of Plasmodium parasites might participate in immune evasion with fibroblasts.In our preliminary study,we found that PvEXP100 was an exported protein of Plasmodium vivax.Furthermore,the effect of PvEXP100 on spleen function will be investigated,which will help to reveal the possible mechanisms of immune evasion of Plasmodium vivax.The main results of this work are listed as follows:(1)Screening of PvEXP100-F1 functional region.The recombinant plasmids pEGFPHSVg-D1-PvEXP100 were constructed,and transfected into 293 T cells.Observed migration of PvEXP100 on the effect of human splenic fibroblasts(HSF)by Transwell experiment.It was found that F1 region could induce a large of HSF migration.To get purified protein,prokaryotic expression recombinant plasmid pET30a-PvEXP100-F1 was constructed and induced expression.(2)Effect of PvEXP100-F1 on HSF.The effect of PvEXP100-F1 on HSF proliferation was observed by CCK-8,and HSF migration was detected by Transwell method.The results showed that PvEXP100-F1 had no effect on HSF proliferation,but could promote HSF migration.The phosphorylation levels of key proteins(ERK,JNK,p38)of MAPK were significantly increased by Western blot,indicating that MAPK was activated by PvEXP100-F1.We found that PvEXP100 could bind HSF by flow cytometry and western blot.It suggested that PvEXP100-F1 may participate in immune evasion of Plasmodium vivax by adsorbing HSF migration and binding to it.In conclusion,PvEXP100-F1 induces HSF migration by activating the MAPK pathway and then binds to it,which may be involved in the immune escape of Plasmodium vivax.Plasmodium infection can promote the polarization of tumor macrophages to M1 type,and inhibit the proliferation,invasion and metastasis of tumor cells.Therefore,the potential application of Plasmodium in tumor immunotherapy was discussed.(1)Effect of PvEXP100-F1 on macrophages.The morphological changes of macrophages were observed by microscopy.Flow cytometry showed that PvEXP100-F1 could polarize macrophages to M1 type.Cell morphology,polarization,NO and cytokine levels were studied by fluorescence microscopy,flow cytometry,and ELISA,respectively.The results suggested that PvEXP100-F1 could significantly activate macrophages and induced bias of macrophages towards M1.The secretion of NO and cytokine of macrophages was increased.The expression levels of iNOS and cytokine mRNAs were elevated by RT-PCR experiment.The effect of PvEXP100-F1 on MAPK expression was studied by western blot.The results showed that PvEXP100-F1 could significantly elevate the expression levels of phospho-MAPK,suggesting that they were activated by PvEXP100-F1.Therefore,PvEXP100-F1 has great immune enhancing acticity effect on macrophages.(2)The direct effect of PvEXP100-F1 on tumor cells.The proliferation and migration of HepG2 cells was detected by CCK-8 and transwell.Both were inhibited.The expression of cyclin D1,anti-apoptotic protein Bcl-2,pro-apoptotic protein Bax and Caspase3 in HepG2 cells was detected by Western blot.PvEXP100-F1 inhibited the expression of Cyclin D1,downregulated the expression of Bcl-2 and up-regulated the expression of Bax and Caspase3.PvEXP100-F1 had directed effct on HepG2 cells.PvEXP100-F1 could polarize macrophages to M1 type,then inducing the phosphyration of MAPK,activating the pathway and elevating the secretion of NO and inflammatory cytokine.Also PvEXP100-F1 can directly inhibit the proliferation and migration of HepG2 and regulate its related cyclins.Therefore,PvEXP100-F1 has potential value in enhancing host anti-tumor innate immunity and tumor immunotherapy,which is worth further study.